One of the distinctive features of the hepatitis B viruses is that infected cells produce multiple types of virus-related particles, including both infectious virions and subviral particles. The principal goals of this program are to define the mechanisms by which subviral particles and virions are formed and to determine how these distinct but related pathways are regulated. To study the assembly of subviral particles we employ two systems: (1) in vitro translation and membrane insertion of S and preS proteins, and (2) transfection of cultured cells with mutant or wild-type S genes. Virion assembly is studied in human hepatoma cell lines transfected with the entire viral genome. Using these systems we will (1) define the transmembrane orientation of the S and preS proteins (2) determine by mutational analysis the regions of each molecule which participate in or regulate the assembly of subviral particles (3) develop an in vitro system for the study of subviral particle assembly (4) explore the possbility of manipulating the pathway of HBsAg particle assembly to generate chimeric particles with vaccine potential and (5) define by mutation the contributions of the individual S and preS proteins to virion formation.